In living plant and animal cells, non-polar lipids are stored in droplets within the cytoplasm and are rarely found in biological membranes. Proteins which have been isolated from membranes of living cells often possess a sequence of non-polar amino acids which anchor the proteins through hydrophobic associations within the interior of phospholipid bilayers. Other proteins are anchored through covalent bonds to glycophospholipids. The oligosaccharide moieties of membrane glycoproteins and glycophospholipids project into the aqueous environment.
Phospholipid monolayers and bilayers form micelles and liposomes which have been used successfully in delivering pharmaceutical agents. However, the chemical and mechanical instability of these constructs have posed problems. Liposomes are prone to oxidation and tend to aggregate and fuse during prolonged storage. Injected liposomes are degraded by the lecithin-cholesterol acyl transferase of high density lipoproteins, and are cleared from the bloodstream by macrophages and hepatocytes. Though it is possible to attach certain glycoproteins to phospholipid micelles, polar phosphate heads facing the aqueous solution tend to inhibit contact of lipid tails with the hydrophobic amino acids of glycoproteins.
Both amino acid sequences and oligosaccharide segments of glycoproteins can contribute chemical and biological properties that may be useful in extracting proteins and glycoproteins from aqueous systems.
Sialic acid, the terminal sugar of may oligosaccharides produced in animal tissue, is ionized at pH 7 (Lehninger et al., 1993). Its presence inhibits uptake and degradation by hepatocytes in circulating blood cells and glycoproteins.
Micelles are aggregates of substances in which hydrophilic polar groups of compounds orient themselves toward and interact with the aqueous phase. The hydrophobic nonpolar hydrocarbon chains of the micelles are hidden within the structure. For example, micelles which contain soap molecules remain evenly suspended in water because their surfaces are negatively charged and the micelles repel each other. Micelles prepared from phospholipids and oligosaccharide-lipid complexes have been used to prepare vaccines using natural and synthetic oligosaccharides, which are immunogens, to prepare stabilized vaccines, as disclosed in U.S. Pat. No. 5,034,519, the entire contents of which are hereby incorporated by reference.
It is also known that amphipathic proteins such as cytochrome oxidase, an intrinsic enzyme found in mitochondrial membrane, when placed in suspension with lipids form sac-like vesicles that are, in effect, man-made membranes. These vesicles have been used as model systems for the study of the isolated protein's relationship with lipid bilayers.
Compans, in U.S. Pat. No. 4,790,987, teaches the preparation of viral glycoprotein subunit vaccines by complexing a lipid with the glycoprotein.
Compans also teaches that the complexes can be obtained by dissolving a lipid in a dialyzable detergent solution containing glycoproteins, then dialyzing the solution to obtain the protein-lipid complex. The lipids are phospholipids. The resulting complexes are then administered in pharmaceutically acceptable carriers.
Rutter et al., in U.S. Pat. No. 4,769,238, note that vaccine bound to a membrane may be superior to non-membrane bound proteins.
Mouritesen et al. have studied protein-protein and protein-lipid interactions in phospholipid bilayers in an attempt to refine the fluid mosaic model of biomembrane proposed in 1972 by Singer-Nicolson. In 1995 Oln et al. produced two-dimensional crystals of avidin on the hydrophobic surface of a phospholipid monolayer. In both synthetic and biological phospholipid membranes, polar heads of component molecules lie adjacent to one another at the aqueous interface, shielding their hydrophobic lipid tails from contact with water or molecules dissolved therein.
In U.S. Pat. No. 5,846,744, Athey et al. describe a novel sensor format based on the impedance analysis of polymer coatings on electrodes for determining the presence or amounts of an analyte in a sample of assay medium.
Organic solvents and detergents have long been used to separate proteins and carbohydrates from the tissues of plants and animals in which they were synthesized. However, deliberate removal from both solvents of a coherent film of glycoproteins fabricated at the oil-water interface is a novel method for separating glycoproteins and their oligosaccharide moieties from solution.
Current methods of glycoprotein recovery and separation often involve the addition of substances which interact chemically with the molecules to be extracted. For instance, dilution with water of soluble glycoproteins in hen egg white causes some components to denature and clump together. In order to facilitate the movement of proteins through gels, filters and other matrices used in the process of protein separation, oligosaccharide moieties are often intentionally removed.
Methods of producing lipo-glycoprotein membranes and micelles are disclosed in Mullen, U.S. Pat. No. 5,824,337, the entire contents of which are hereby incorporated by reference. Lipophilic amino acids of glycoproteins adhere through non-covalent hydrophobic interactions to molecules of oil, while hydrophilic moieties form hydrogen bonds with water molecules in the aqueous phase. In addition, proteins may adhere to one another in the plane of the oil-water interface through a variety of non-covalent associations, including sulfur bonds, to produce a membrane that is mechanically and chemically stable relative to a phospholipid membrane.
Carbohydrate moieties of glycoproteins are key components of various intercellular recognition processes. Glycoproteins on the outer surface of the micelle can be chosen to target appropriate tissue, inhibit uptake by tissue, and induce endocytosis of the micelle. Antibodies, apoproteins, and opsonins are examples of glycoproteins that mediate these responses.